Schematics of DNA transgenes. A, The first target DNA, QC288A329A, contains the constitutive promoter SCP1 driving a mutant ALS gene. A FRT1 site (black triangle) is placed between the SCP1 promoter and the ALS gene, and a FRT87 site (white triangle) is placed at the 3′ end. B, Predicted first RMCE DNA, QC288A436A, from the transformation of QC288A329A target with the first donor, QC436. All the components between the FRT1 and FRT87 sites of QC288A329A are replaced by the components between the FRT1 and FRT87 sites of QC436. The third recombination site, FRT12 (black and white triangle), is introduced. C, Predicted second RMCE DNA, QC288A436A438A, from the retransformation of the first RMCE DNA, QC288A436A, now used as the second target, with the second donor QC438. The components between the FRT1 and FRT12 sites of QC288A436A are replaced by the components between the FRT1 and FRT12 sites of QC438. Relative positions of qPCR assays (vertical arrows), PCR primers, and MfeI-cleavage sites are marked. Black bars represent Southern hybridization probes specific to SCP1, HPT, FATB-2, and DGAT1. FLP QC292 and predicted excision DNA QC288ME, which lost all the components between the FRT1 and FRT87 sites, were described previously (Li et al., 2009).

Analysis of the first-round SSI events. PCR assays specific to the genomic borders of the B target site hosting different transgenes was done using combinations of the 5′ border, 3′ border, and transgene-specific primers (Fig. 2). A, Expected PCR fragments of the 5′ border (left), 3′ border (center), and excision QC288ME (right) of the first RMCE, QC288A436A, are 886, 1,116, and 986 bp. The expected 9,108-bp-long full-length QC288A436A is too large to be amplified (right). B, Expected PCR fragments of the 5′ border (left) and 3′ border (center) of target QC288A329A and the FRT12 region of RMCE QC288A436A (right) are 967, 1,180, and 693 bp. Wild-type DNA (wt) was included as a negative control. The four events were all chimeras containing the RMCE, target, and excision transgenes at the embryogenic callus stage. The FlashGel DNA markers are 4, 2, 1.25, 0.8, 0.5, 0.3, 0.2, and 0.1 kb (Lonza Rockland).

Analysis of the second round RMCE event. PCR assays specific to the genomic borders and internal regions of the second RMCE DNA, QC288A436A438A, were done on RMCE T0 plant B531-1 using various primer combinations (Fig. 2). The hemizygous (RMCE/excision) B531-1 ancestors hemizygous B53, homozygous B5 and B, and wild-type DNA (wt) were included as controls. A, The expected 886-bp 5′ border of both QC288A in B and QC288A436A in B53 (left) and the 561-bp 3′ border of QC288A in B (center) were amplified. The full-length 4,742-bp QC288A of B, 6,331-bp QC288A329A of B5, and 986-bp QC288ME (excision) of B53 and B531 were amplified (right). The expected full-length 9,108-bp QC288A436A of B53 and 21,925-bp QC288A436A438A of B531-1 were too large to be amplified. B, The expected 967-bp 5′ border of both QC288A329A in B5 and QC288A436A438A in B531 (left) and the 1,180-bp 3′ border of QC288A329A in B5 (center) were amplified. The same B5 band was also amplified from B53 that contained chimerically B5 DNA QC288A329A. The expected 1,116-bp 3′ border of both QC288A436A in B53 and QC288A436A438A in B531-1 were amplified (right). C, A 693-bp fragment unique to the FRT12 region of QC288A436A was amplified only in B53 (left). An 840-bp fragment unique to the FRT12 region (center) and a 711-bp fragment unique to the 5′ region of QC288A436A438A were amplified only in B531-1 (right). The FlashGel DNA markers are 4, 2, 1.25, 0.8, 0.5, 0.3, 0.2, and 0.1 kb. D, A 2,946-bp segment covering the DHPS, MYB2 terminator, and UBIQ10 promoter of QC288A436A438A was lost in T0 plant B531-1 although intact in embryogenic callus B531. The expected 5,753-bp Ph3-1/CRS-10 band (left) and 9,216-bp Dgat-1R/CRS-10 band (right) of intact QC288A436A438A were amplified from the embryogenic callus B531 and QC438 donor DNA but not from the T0 plant B531-1 leaf DNA, which produced approximately 3-kb smaller bands. Some nonspecific bands were amplified by the long-range PCR. The 1-kb DNA markers are 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, and 1 kb (Invitrogen).

Confirmation of gene stacking by Southern hybridization. Genomic DNA of B53 T1 plants, homozygous RMCE B53-1 and B53-2, hemizygous (RMCE/excision) B53-3 and B53-4, and excision B53-5 and B53-6, the T0 plant B531-1 (RMCE/excision), and homozygous B5 and B ancestor plants were digested with MfeI and hybridized sequentially with probes SCP1, HPT, FATB-5, and DGAT1. The expected sizes of Southern bands are specific to transgenes integrated at the B target site, where the transgenic QC288A lost 17-bp 5′ end and 49-bp 3′ end sequences. There is an MfeI site 2,131 bp upstream and 1,230 bp downstream, respectively, of the transgenes that contain MfeI sites (Fig. 2). A, The SCP1 probe hybridized to the expected 12,205-bp band of QC288A436A in B53-1 and B53-2, the 3,987-bp band of QC288ME in B53-5 and B53-6, and both bands in hemizygous plants B53-3 and B53-4. The same 3,987-bp QC288ME band and a 3,681-bp QC288A436A438A band were hybridized in B531-1. As expected, the same 3,681-bp band also specific to QC288A329A was detected in B5 and a 7,839-bp QC288A band was detected in B. B, The HPT probe hybridized to the same 12,205-bp QC288A436A band in B53-1, B53-2, B53-3, and B53-4 and to the same 7,839-bp QC288A band in B. C, The FATB-5 probe hybridized to two endogenous bands in all samples in addition to the same 12,205-bp QC288A436A band in B53-1, B53-2, B53-3, and B53-4. Instead of the expected 12,931-bp band of QC288A436A438A, a larger band, likely the 16,402-bp band expected from modified QC288A436A438A with its 2,946-bp DHPS-MYB2-UBIQ10 deleted, overlapped with the top wild-type band (wt) in B531-1. D, The DGAT1 probe detected in B531-1 the same 16,402-bp band of the modified QC288A436A438A instead of an expected 6,352-bp QC288A436A438A band. The DIGVII DNA markers are 8,576, 7,427, 6,106, 4,899, and 3,639 bp (Roche).

↵a Events B51 to B54 are representative hygromycin-resistant events selected from the retransformation of homozygous target QC288A329A with the first donor QC436 and FLP QC292 DNA. B53-1 to B53-6 are segregating T1 plants derived from event B53.

↵b Embryogenic callus or T1 plant leaf samples were analyzed by qPCR specific to the SCP1 and HPT junction of QC288A436A (SSI), a QC436-specific region (Donor), the SCP1 and ALS junction of QC288A329A (Target), and a QC292-specific region (FLP). A heat shock protein gene, HSP, was used as an endogenous control in duplex qPCR. A genomic DNA sample containing one copy of the respective transgene was used as the calibrator to calculate relative transgene copies in other samples using the Applied Biosystems 7500 system software. A value of less than 0.3 or between 0.4 and 1.3 was considered as zero or one copy. A value between 1.4 and 2.3 was considered as two copies.

↵c Fatty acids were determined on the bulk of 10 mature somatic embryos for B51 to B54 or on T1 seed chips for B53 by GC and expressed as the percentage of total fatty acids. Fatty acid measurements by GC as described here are reproducible to approximately 3% of total fatty acids. Untransformed control somatic embryos typically contain 12.6% to 20.8% 16:0, 4.2% to 6.6% 18:0, 12.3% to 22.9% 18:1, 39.3% to 46.9% 18:2, and 12.4% to 23.5% 18:3 (Meyer et al., 2008). Increases in 18:1 to greater than 30% and decreases in 16:0 to less than 10% are indicative of successful FAD2 and FATB gene down-regulation, respectively. The chipped B53 seeds later germinated to T1 plants that provided leaf DNA for the qPCR assay.

↵a Events B531 to B512 are representative chlorsulfuron-resistant events selected from the retransformation of the embryogenic callus of RMCE events B51, B53, and B54 containing QC288A436A with the second donor QC438 and FLP QC292 DNA.

↵b Embryogenic callus samples were analyzed by qPCR specific to the SCP1 and ALS junction of QC288A436A438A (SSI), a QC438-specific region (Donor), the SCP1 and HPT junction of QC288A436A (Target), and a QC292-specific region (FLP). The qPCR assays were done as described in the Table I legend.

↵c Fatty acids in mature somatic embryos were determined by GC as described in the Table I legend and are expressed as the percentages of total fatty acids.

↵d Oil contents in mature somatic embryos determined by NMR are presented as the percentages of total sample dry weight. Oil measurements made by NMR as described here are reproducible to approximately 1% oil of sample dry weight. Untransformed control somatic embryos typically contain 2.2% to 6.2% oil (Meyer et al., 2008). Increases in oil contents to above 7% oil of sample dry weight are indicative of a functional DGAT1 gene. Oil content for event B532 was not determined (ND) due to low tissue amounts.